Studies from the last several years have repeatedly highlighted the importance of long noncoding RNAs (lncRNA) in epigenetic regulation, development, and disease. However, the mechanisms by which RNAs control these processes remain poorly defined. Improved understanding of RNA-based mechanisms is crucial, especially given that 70-80% of the mammalian genome is transcribed and that the vast bulk of transcription is noncoding. Nowhere is the abundance of lncRNAs more evident than at the X-inactivation center (Xic), an X-linked region that controls the X-chromosome inactivation (XCI) in the female mammal. XCI serves as an excellent model to study lncRNA regulation because this process is controlled by a series of RNA-based switches. Silencing is initiated by the 17-kb Xist RNA as it recruits Polycomb proteins to the X-chromosome. Xist RNA is in turned controlled by an antisense transcript, Tsix, which antagonizes Xist by repelling the recruitment of an RNA-protein complex containing Polycomb proteins. My laboratory has also found that Xist upregulation requires the action of two additional lncRNAs, RepA which recruits Polycomb proteins to the Xic, and Jpx which is required to activate Xist transcription. This RO1 grant has, for the past 13 years, provided crucial support for this lncRNA research. Herein, we propose to further advance our understanding of RNA regulation by studying how RNAs of the Xic control the different steps and forms of XCI. Specifically, we will: (i) Investigate whether and how RNAs play a role in homologous X-chromosome pairing, a process proposed to regulate counting and choosing of X-chromosomes for inactivation; (ii) Determine how Xist RNA is turned on by Jpx RNA; and (iii) Elucidate fundamental differences between random and imprinted XCI, which our research has shown to be differentially dependent on Xist RNA for silencing. That the proposed work will require five years to complete and is expected to train five postdoctoral fellows for academic careers in the fields of epigenetics, RNA regulation, and stem cell biology. PUBLIC HEALTH RELEVANCE: Studies from the last several years have highlighted the importance of long noncoding RNAs in human development, health, and diseases such as cancer and atherosclerosis, but the mechanisms by which RNAs effect epigenetic change remain largely unknown. X-chromosome inactivation serves as an excellent model to study RNA regulation because this process is controlled by a series of RNA-based switches. Our proposed research will dissect mechanisms of RNA regulation by investigating its role in chromosome pairing, gene silencing, and imprinting.